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Genetic Elements that spread multi-drug resistance
determinants in Gram-negative bacteria:
how complex is the problem?
Timothy R. Walsh
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Remit
of
Talk
• Will
not
detail
integrons
and
movement
of
integrons
ES
C
M
ID
O
• Will
choose
few
and
only
key
examples
of
1.
plasmids
2.
transposons
3.
ISCR
elements......
to
illustrate
clinically
import
examples
–
and
choose
key
resistant
genes
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Channel Four Study
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171 swabs
-156 grew meropenem resistant
Gram-negatives)
- 51/171 (29.8%) were positive for
NDM-1
50 water samples
- 14 grew meropenem resistant
Gram-negatives)
- 2 out of 50 (4%) were positive for
NDM-1
Environmental positive samples
C
M
ID
O
Escherichia coli
Suttonella indologenes
Shigella boydii
Stenotrophomonas maltophilia
Aeromonas caviae
Citrobacter freundii
Pseudomonas oryzihabitans
Pseudomonas pseudoalcaligenes
Pseudomonas putida
Vibrio cholera
ES
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116
97
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3
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O
ID
M
C
ES
Walsh et al., Lancet Infectious Diseases, April 2011
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O
ID
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ES
Walsh et al., Lancet Infectious Diseases, April 2011
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blaNDM-1 genes are carried on AC plasmids
Some isolates have two copies of blaNDM-1
P
T
P
T
P
T
P
O
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ID
M
C
ES
T
T
M
P
T
P
T
P
T
P
ib
ra
ry
–IR9
IR9
IR19
IR22
–180kb
–P T P T P T P T
O
–160kb
–150kb
–IR3
IR9
IR19
IR22
n
© lin
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–P T P T P T P T
ib
ra
ry
–Changes
to
NDM‐1
A/C
plasmids
during
conjuga;on
ES
C
M
ID
–90kb
–F
–A/C
ry
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What are Transposons?
Transposons, also called "hopping genes" are
segments of DNA that are able to move around in
the genome
ID
O
They were originally suggested by Barbara
McClintock based on research she did the 1930's
and 1940's
ES
C
M
The simplest kind code only for the enzymes that
cut them out of their current DNA molecule and
insert them into another one
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What
are
IS
Elements
ES
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M
ID
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• Small
and
simple.....generally
encode
no
funcCons
other
than
those
involved
in
their
mobility
• Required
in
cis,
in
par,cular
recombina,onally
ac,ve
• DNA
sequences
which
define
the
ends
of
the
element,
together
with
an
enzyme,
the
transposase
which
recognizes
and
processes
these
ends
• The
transposase
is
generally
encoded
by
one
or
perhaps
two
open
reading
frames
and
consumes
nearly
the
enCre
length
of
the
element
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Mechanism of transposition
ID
O
• The simplest sequence of events:
• The transposase recognizes and binds to the transposable
element
• It then acts as a site-specific endonuclease, cleaving the DNA to
expose the element's 3'-OH ends
• A duplex target DNA then binds to the complex
• Direct attack of the 3'-OH ends on phosphates of the duplex
cleaving it and joining it to the transposon
• Since the phosphates attacked are staggered, short single
strand gaps remain, which are filled in by the regular repair
mechanisms
ES
C
M
• Mode of binding is the same as the mode of binding of
transcription factors to DNA: a helix in the major groove.
• Some Type 1 transposases cleave the 5' ends of the transposon
as well.
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ES
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Tn5
Bound
to
Ends
of
Transposon
DNA
Binding
Domain
of
Tn3
Transposase
Bound
to
the
Transposon
ry
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Nomenclature of Transposons
ES
C
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Revised
nomenclature
for
transposable
gene,c
elements
Chandler et al., 2008. Plasmid, 60; 167-173
ry
INT
aacA7
blaVIM‐2
blaVIM‐2
aacA4
ES
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M
ID
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INT
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GeneCc
Elements
changing
and
moving
resistance
genes
around
Europe
aacC1
aacA4
qacEÄ1/sul
French VIM-2
qacEÄ1/sul
Poland VIM-2
ES
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Schematic representation of the blaIMP-13 and blaVIM-2 genetic loci, and comparison with
the 5′ end of the composite mercury resistance transposon Tn21, which includes the
transposition (tnp) and integron In2 region (GenBank accession no.
Toleman M A et al. J. Antimicrob. Chemother. 2003;52:583-590
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Tn5090/Tn402‐like
IRi
aadA2
aadB
IRt
qacEΔ1/sul
Δtnib
tniA
ES
C
M
ID
O
TGTCGTTTTCAGAAGACGGCTGCAC-------AGGGGTAGTGAATCCGCCAGATTGACTTGCGCTGCCCTACCTCTCACTAGTGAGGGG
TGTCGTTTTCAGAAGACGGCTGCAC-------AGGGGTAGTGAATCCGCCAGATTGACTTGCGCTGCCCTACCTCTCACTAGTGAGGGG
TGTCGTTTTCAGAAGACGGCTGCAC-------AGGGGTAGTGAATCCGCCAGATTGACTTGCGCTGCCCTACCTCTCACTAGTGAGGGG
TGTCGTTTTCAGAAGACGGCTGCAC-------AGGGGTAGTGAATCCGCCAGATTGACTTGCGCTGCCCTACCTCTCACTAGTGAGGGG
TGTCGTTTTCAGAAGACGGCTGCAC-------AGGGGTAGTGAATCCGCCAGATTGACTTGCGCTGCCCTACCTCTCACTAGTGAGGGG
TGTCGTTTTCAGAAGACGGCTGCAC-------AGGGGTAGTGAATCCGCCAGATTGACTTGCGCTGCCCTACCTCTCACTAGTGAGGGG
TGTCGTTTTCAGAAGACGGCTGCAC-------AGGGGTAGTGAATCCGCCAGATTGACTTGCGCTGCCCTACCTCTCACTAGTGAGGGG
TGTCGTTTTCAGAAGACGGCTGCAC-------AGGGGTAGTGAATCCGCCAGATTGACTTGCGCTGCCCTACCTCTCACTAGTGAGGGG
TGTCGTTTTCAGAAGACGGCTGCAC-------AGGGGTAGTGAATCCGCCAGATTGACTTGCGCTGCCCTACCTCTCACTAGTGAGGGG
TGTCGTTTTCAGAAGACGGCTGCAC-------AGGGGTAGTGAATCCGCCAGATTGACTTGCGCTGCCCTACCTCTCACTAGTGAGGGG
TGTCGTTTTCAGAAGACGGCTGCAC-------AGGGGTAGTGAATCCGCCAGATTGACTTGCGCTGCCCTACCTCTCACTAGTGAGGGG
TGTCGTTTTCAGAAGACGGCTGCAC-------AGGGGTAGTGAATCCGCCAGATTGACTTGCGCTGCCCTACCTCTCACTAGTGAGGGG
IRi
*
integrase
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ES
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Evolution of Tn5090/Tn402-like elements
Toleman et al., 2007. Antimicrob Agents Chemother. 51 2636-8.
ry
-3 and the PCR scheme used to amplify it.
C
M
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Representation of Tn4401a harbouring blaKPC
ES
Curiao T et al. J. Antimicrob. Chemother. 2010;65:1608
-1614
© TheAuthor 2010. Publishedby OxfordUniversity Press on behalf of theBritishSociety for
Antimicrobial Chemotherapy. All rights reserved. For Permissions, pleasee
-mail:
[email protected]
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ES
Tato et al., 2010. ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 54; 320–327
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ID
M
C
ES
Tato et al., 2010. ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 54; 320–327
ry
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O
ID
M
C
ES
Lar,gue
et
al.,
2004.
FEMS.
234:
201‐207
ES
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M
ID
O
n
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ry
ISEcp1B
mediated
transposiCon
Poirel et al., 2005. AAC. 49:447-550
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What
are
ISCR
Elements?
ES
C
M
ID
O
• One
ended
transposiCon
elements
• Only
recently
been
associated
with
anCbioCc
resistance
• Replicate
via
rolling
circle
transposiCon
• Capable
of
carry
large
segments
of
DNA
to
their
leX
hand
side
including
many
anCbioCc
resistance
genes
ry
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Common Regions - 1993
catA2 qac/sul orf5/orf6
aadA2 qac/sul
aadB qac/sul
AJ517791
ID
O
dfrA10 qac/sul orf5
Common
Region
ES
C
M
L06822
(In6)
Stokes HW, Tomaras C, Parsons Y, Hall RM. Plasmid. 1993, 30:39-50
L06418
(In7)
blaOXA-45
terIS
eltA/B
terIS
ES
C
M
ID
O
co‐transposed
genes
n
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ry
ib
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Direc&on
of
rolling
circle
replica&on
ISCR5
oriIS
IS91
oriIS
ry
ib
ra
FormaCon
of
Complex
Class
1
integrons
aadA2
qacEΔ1/sul
orf5/orf6
ISCR1
Deletion event removing terIS
and fusing the ISCR1 element
to the 3’ conserved sequence of
the class 1 integron
C
aadA2
ES
intI1
oriIS
M
ID
O
intI1
qacEΔ1/sul
oriIS
n
© lin
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terIS
ISCR1
ry
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© lin
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O
ID
M
C
ES
Coque
and
Canton.
Current
Opinion
in
Microbiology.
2006.
oriIS
intI1 aadA2 qacEΔ1/sul1
ry
ISCR1
(ii)
(iii)
(iv)
dfrA10 qacEΔ1/sul1
(ii)
ISCR1
(iii)
dfrA10
intI1 aadA2 qacEΔ1/sul1
ISCR1
ISCR1
(v)
dfrA10 aadA2 qacEΔ1/sul1
(vi)
dfrA10
intI1 aadA2 qacEΔ1/sul1
ISCR1
ISCR1
ISCR1
A
aadB
dfrA10 aadA2 qacEΔ1/sul1
n
© lin
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(i)
dfrA10 qacEΔ1/sul1
ib
ra
(i)
C
B
aadB
orf5/orf6
qacEΔ1/sul1
orf5/orf6
aadB
aadB
ID
A
O
qacEΔ1/sul1
aadB
dfrA10
ISCR1
qacEΔ1/sul1
ISCR1
orf5/orf6
aadA2 dfrA10
orf5/orf6
ES
C
M
B
ISCR1
orf5/orf6
Toleman
et
al.,
JAC.
2006
C
aadB
ISCR1
aadA2 dfrA10
orf5/orf6
ISCR1
ry
ib
ra
n
© lin
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O
ID
M
C
ES
Toleman
et
al.
2007.
Emerging
Infec,ous
Diseases.
13;
559‐565
qac/sul
floR
qac ISCR3
tetG
qac/sul orf5
intI1/groEL
ib
ra
aadA2
ry
Importance of ISCR3 elements (55%ID ISCR1)
blaPSE‐1
aadA2 qac/sul
floR
tetG
qac/sul
intI1/groEL
qac
dfrA10 qac/sul orf5
aadA2
n
© lin
by e
L
au ec
th tu
or re
L
blaPSE‐1
aadA2 qac/sul
qac/sul
orf5/6 qac/sul
floR
dfrA1 orfC qac/sul
intI1/groEL
qac
tetG
blaPSE‐1
floR
tetG
qac
IS6100
intI1/groEL
SGI1‐D
floR
SGI1‐E
qac/sul
blaPSE‐1
SGI1‐F
hhp://www.cardiff.ac.uk/medic/aboutus/departments/medical
floR
tetG qac
intI1/groEL
orfC qac/sul orf5/6
SGI1‐I
microbiology/geneCcs/iscrelements.html
dfrA1
qac/sul
dfrA1 orfC
SGI1‐A
dfrA10 qac/sul orf5
IS6100
aadA2
SGI1
floR
qac/sul
tetG
qac
O
yieF/yieG
ES
Scale
=1kb
SGI1‐J
AY434092
AY434093
yieE/yieF/orfX
C
M
ID
yieE
intI1/groEL qac/sul
blaTEM‐1 rmtB
AY434091
erm
A15097
intI1/groEL
tnpR
Toleman
et
al.
2006.
Microbiology
and
Molecular
Biology
Reviews.
70:
296‐316.
AB103506
ib
ra
ry
Integrating conjugative elements (ICEs)
n
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•A class of bacterial mobile genetic elements that disseminate via conjugation and then
integrate into the host cell genome.
•The SXT/R391 family of ICEs consists of more than 30 different elements that all
share the same integration site in the host chromosome but often encode distinct
properties.
•Comparative analyses of the genomes of several SXT/R391 ICEs, found that the
genomes have been shaped by inter–ICE recombination.
M
ID
O
•Conjugation facilitates the segregation of hybrids and could provide a means to select
for functional recombinant ICEs containing novel combinations of genes conferring
resistance to antibiotics.
ES
C
•ICEs promote their own diversity and can yield novel mobile elements capable of
disseminating new combinations of antibiotic resistance genes.
Genevie`ve
et
al.,
2009.
PlosGen
5;
1‐11
O
n
© lin
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ID
M
C
ES
ib
ra
How
complex?
ry
ry
ib
ra
n
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O
ID
M
C
ES
Fournier
et
al.
2006.
Compara,ve
genomics
of
mul,‐drug
resistance
in
Acinetobacter
baumannii.
Gene,cs.
2:
62‐72
ES
C
M
ID
O
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ra
ry
AnCbioCc
genes
belonging
to
MDR
A.
baumannii
strain
AYE
Fournier
et
al.
2006.
Compara,ve
genomics
of
mul,‐drug
resistance
in
Acinetobacter
baumannii.
Gene,cs.
2:
62‐72
blaNDM-1
ΔIS26
ry
ΔblaDHA-1 ΔPAI
ΔTn3
ib
ra
Δldh
n
© lin
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or re
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efflux pump
(4-6kb)
Int1
arr-2
ereC
aadA1
cmlA7
qacEΔ1
ES
C
M
ID
O
(1-4kb)
Yong et al., 2009. Antimicrobial Agents and Chemotherapy. 53; 5046-54
Poirel et al. 2010. Antimicrobial Agents and Chemotherapy. 54; 4914-4916
ISCR1
ib
ra
Acinetobacter spp.
AbaI1
25
NDM-1
Enterobacteriaceae
Gene X
∆AbaI1
25
..................................................................
ID
NDM-1
ISCR
groEL
groES
ES
C
M
ISCR
NDM-1
Curr
Opin
Microbiol.
2010
Dec;13(6):781‐5.
Toxin‐an,toxin
systems:
why
so
many,
what
for?
Van
Melderen
L.
O
Enterobacteriaceae
..................................................................
n
© lin
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Gene X
ry
The perfect weapon?
DHA-1, CMY-6, aar-2, ereC, aadA1, aacA4, cmlA7, armA, rmtC, rmtD, sul1,
dhfr, sul2, toxin-anti-toxin system - MqsA
ry
ID
O
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Chronology
of
movement
of
AbxR
genes
Event
3
Event
4
C
ES
Event
2
M
Event
1
ry
ES
C
M
ID
O
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ra
Major structural features of pKOX105, encoding VIM
-1, QnrS1 and SHV -12, in comparison with
IncN plasmids p9 and p12 carrying KPC -2 and KPC -3, respectively, and the IncN reference
plasmid R46.
Carattoli A et al. J. Antimicrob. Chemother. 2010;jac.dkq269
© TheAuthor 2010. Publishedby OxfordUniversity Press on behalf of theBritishSociety for
Antimicrobial Chemotherapy. All rights reserved. For Permissions, pleasee
-mail:
[email protected]
ry
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ib
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How complex is the problem?
ES
C
M
ID
O
• Although
known
for
a
long,
transposons
have
only
recently
been
associated
with
MDR
and
PDR
Gram‐
negaCve
bacteria
• Safe
haven
for
inserCon
but
can
also
be
destrucCve
• This
associaCon
may
or
may
not
be
associated
with
drug
consumpCon
but
anCbioCcs
such
as
fluoroquinolones
have
secondary
effects
promoCng
gene
mobility
• The
complexity
(and
associated
plasCcity
and
fluidity)
will
become
greater
over
Cme
• A
warning
to
genome
sequencers
O
n
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ID
M
C
ES
ib
ra
ry
• Jan Bell
C
M
ID
O
Mark Toleman
Dongeun Yong
Sonia Ferreira
Allaaeddin El Salabi
Sahim Agouri
Jonathan Tynddell
Janis Weeks
Mandy Wootton
Robin Howe
Vicky Davies
ES
•
•
•
•
•
•
•
•
•
•
n
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Acknowledgements
• David Paterson
• Hanna Sdajbat
• Laurent Poirel
• Patrice Nordmann